Transmissible spongiform encephalopathies (TSEs) are a group of fatal and infectiousneurodegenerative disorders affecting humans and animals. Although rare, the recent outbreak of Bovine Spongiform Encephalopathy and Chronic Wasting disease and the transmission of the disease from cattle to humans have caused great concern about a possible epidemic in humans. This problem is aggravated by the lack of an efficient, sensitive and early diagnosis as well as many uncertainties surrounding the unprecedented nature of the infectious agent, its mechanism of propagation and the species-barrier that controls prion transmission. The most accepted hypothesis proposes that the infectious agent (termed prion) is composed exclusively by a misfolded version of the normal prion protein (termed PrPSc), and the disease is transmitted by propagation of the misfolding from the disease associated isoform to the normal host protein (termed PrPc). We have recently described a procedure to induce the conversion of PrPc into PrPSc in vitro starting with minute quantities of brain PrPSc. This procedure, named Protein Misfolding Cyclic Amplification (PMCA) mimics the process of prion replication in vivo, but at an accelerated speed resulting in an exponential amplification of the initial amount of PrPSc. The major goal of this Project is to study the replication of human prions in vitro using PMCA and to develop a sensitive, non-invasive diagnostic tool for human TSEs in biological fluids or peripheral tissues. In specific aim 1 we will attempt to develop a highly sensitive diagnostic tool for human TSE, by detecting PrPSc in fluids (blood, CSF or urine) or peripheral tissues (muscle). In specific aim 2 we will study and quantify the species barrier for the transmission of prionsfrom diverse species into human. Specific aim 3 proposes to assess the competence of unconventional formsof human PrPSc to convert human PrPc by PMCA amplification. In specific aim 4 we will study the size distribution of PrPSc from diverse human prion strains and their ability to sustain prion replication in vitro. This project offers a balanced combination between basic science studies aimed to understand the most relevant scientific problems in the human prion field and applied studies to resolve the main practical problem associated to these diseases, which is the lack of a highly-sensitive pre-symptomatic diagnosis to limit the spreading of these incurable illnesses.